Project Summary Despite the existence of vaccines and antiviral drugs, global annual death tolls attributed to influenza virus infection are ~500,000. Currently there is only one orally bioavailable drug, oseltamivir, that is still in use to treat influenza infection. The alarming fact is that oseltamivir-resistant influenza strains have already been isolated from human patients, and several of them appear to have adapted the fitness of transmission. Thus there is a pressing need to develop novel antivirals to combat these drug-resistant influenza viruses. They can be used either alone to inhibit oseltamivir-resistant strains or in combination with oseltamivir to decrease the pace of drug resistance evolution. Moreover, as the influenza viruses circulating among humans consist of at least two influenza A strains (H1N1 and H3N2) and two B strains (Yamagata and Victoria), it is also desirable to have one antiviral drug with broad-spectrum antiviral activity against all four strains. In response to the need for a next generation of antiviral drugs with broad-spectrum antiviral activity, especially against multidrug-resistant influenza viruses, we performed an in silico screening of an in-house library of small molecules predominantly prepared by one-pot multicomponent reaction (MCR) methodologies against the virus polymerase PA subunit and have identified several promising hits. One compound, UAWJ85, inhibits several multidrug-resistant influenza A and B viruses with EC50 values range from single to sub-micromolar. This compound also displays a high in vitro genetic barrier to drug resistance, as no resistant viruses were selected after 10 passages with increasing concentrations of the compound. Mechanistic studies confirmed the inhibition of polymerase PA-PB1 subunit interactions by UAWJ85. The broad-spectrum antiviral activity and high in vitro genetic barrier to drug resistance of UAWJ85, coupled with the expeditious structure?activity relationship studies using the one-pot Ugi- Azide 4CR methodology, have led us to further optimize the antiviral potency, selectivity index, in vitro and in vivo PK properties of lead compounds and test their in vivo antiviral efficacy in mice. In Aim 1, we will optimize the in vitro antiviral and pharmacokinetic properties of UAWJ85. A list of criteria was imposed for compound progression. The goal is to prioritize lead compounds for in vivo mice studies. In Aim 2, we will will test the in vivo PK and antiviral activity of UAWJ85 or its analogs using the influenza virus infected mice model. In summary, the advantage of exploring MCR products for broad-spectrum anti-influenza drugs has been clearly demonstrated by the preliminary results. This proposal, if successfully implemented, will lead to the urgently needed antivirals to combat both seasonal outbreaks and the next influenza pandemic.